The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels  T. Hao, N. Wen, J.-K. Cao, H.-B. Wang, S.-H. Lü, T. Liu, Q.-X. Lin, C.-M. Duan, C.-Y. Wang  Osteoarthritis and Cartilage  Volume 18, Issue 2, Pages 257-265 (February 2010) DOI: 10.1016/j.joca.2009.08.007 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Formulation of chitosan hydrogel and reconstruction of tissue-engineered cartilage in vitro. A, In room temperature, the chitosan hydrogels were keep in liquid; B, The chitosan hydrogels kept at 37°C in 10–15min were solidified; C, The reconstructed cartilage in the 4-well plate; D, The AO/PI staining showed that the chondrocytes remained >90% viable in the solid chitosan matrix after cultured 1 day in vitro, AO: green; PI: red. Bar=0.5cm(C); 100μm (D). Osteoarthritis and Cartilage 2010 18, 257-265DOI: (10.1016/j.joca.2009.08.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Accumulation of matrix in the tissue-engineered cartilage. Histology and immunohistochemistry of chondrocytes cultured in chitosan hydrogels 3 weeks. The results showed that the chondrocytes in the chitosan hydrogels accumulated pericellular sulfated GAG-containing matrix. A, H.E. staining; B, Type II collagen immunohistochemical staining; C, Toluidine blue staining; D, Safranin O staining. Star: chitosan hydrogel; Arrowhead: cell nucleus; Arrow: matrix of the chondrocytes. Bar=100μm. Osteoarthritis and Cartilage 2010 18, 257-265DOI: (10.1016/j.joca.2009.08.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Gross observation of the articular cartilage repair at 24 weeks post-operation. A, the defect part of the cartilage in the experimental group was covered by the smooth, consistent, glistening white hyaline tissue nearly indistinguishable from the surrounding normal cartilage. No clear signs of margin with normal cartilage could be spotted on the surface of the regenerated areas; B, The defects in control group 1 were partially repaired with fiber-like tissue, leaving a small depression in the defect areas; C, The defects in control group 2 detected a thin and irregular surface tissue, with obvious defects and cracks surrounding the normal cartilage. Arrow: the defect; Bar=0.5cm. Osteoarthritis and Cartilage 2010 18, 257-265DOI: (10.1016/j.joca.2009.08.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Histological evaluation of the articular cartilage repair at 12 weeks post-operation. (A–C), In experimental group, H.E. staining displayed that the boundary of normal cartilage and renewed cartilage. The cells in the renewed cartilage were somewhat different from those in the normal cartilage tissue (A); The formation of cartilage-like tissue could be seen with toluidine blue (B) staining and safranin O staining (C), indicating a considerable amount of proteoglycan; (D–F), In control group 1, the defect areas were filled with the remaining chitosan hydrogels materials surrounded by fibrocartilage tissues (D). It seemed that no cartilage-like tissue was formed as shown by toluidine blue (E) staining and safranin O staining (F); (G–I), In control group 2, H.E. staining showed that there were no typical cells (G), the negatively toluidine blue staining indicated that no cartilage tissue (H), but only a small quantity of fibrous tissues could be seen along the margin of defects (I). Dotted line indicated the boundary of the normal cartilage and the renewed cartilage. Bar=100μm. Osteoarthritis and Cartilage 2010 18, 257-265DOI: (10.1016/j.joca.2009.08.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Histological and immunohistochemical evaluation of the articular cartilage repair at 24 weeks post-operation. (A–D), In experimental group, H.E. staining showed that newly-formed cartilage became more mature. The cells in the newly cartilage were similar to the normal chondrocytes (A). The regenerated hyaline-like cartilage had less intensive toluidine blue staining than adjacent normal cartilage (B). A typical structure of hyaline cartilage lacunae is apparent in the regenerated area (C). The regenerated surface was demonstrated to be hyaline-like by type II collagen immunohistochemical staining (D); (E–H), In control group 1, no chitosan hydrogels were observed (E). The defects were partly filled with some renewed tissues when modestly stained with toluidine blue (F) and safranin O/fast green (G). Type II collagen immunohistochemical staining showed the cartilage to be fibrocartilage other than hyaline-like cartilage (H); (I–L), In control group 2, H.E. staining indicated that the defect areas have no cells existence (I). The negative staining by toluidine blue (J), safranin O/fast green (K) showed that the cartilage defects containing only some loose fibrous tissues. Type II collagen immunohistochemical staining contained the same (L). Dotted line indicated the boundary of the normal cartilage and the renewed cartilage. Bar=100μm. Osteoarthritis and Cartilage 2010 18, 257-265DOI: (10.1016/j.joca.2009.08.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions